Modified cytotoxins and their therapeutic use

ABSTRACT

The present disclosure generally provides compounds useful for treating cancer. In some aspects, the disclosure provides small-molecule cytotoxins that are chemically modified to include one or more moieties that include hydrophobic portions. In some embodiments, the disclosure provides small-molecule cytotoxins that are chemically modified with fatty acid-containing moieties. In some aspects, the disclosure provides compositions, such as pharmaceutical compositions, that include such modified small-molecule cytotoxins and a protein. In some embodiments, the protein is albumin or an albumin mimetic. Further, the disclosure provides various uses of these compounds and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S.patent application Ser. No. 15/271,822, filed Sep. 21, 2016, whichclaims the benefit of priority of U.S. Provisional Application No.62/222,059, filed Sep. 22, 2015, both of which are hereby incorporatedby reference as though set forth herein in their entirety.

TECHNICAL FIELD

The present disclosure generally provides compounds useful for treatingcancer. In some aspects, the disclosure provides small-moleculecytotoxins that are chemically modified to have one or more moietiesthat include hydrophobic portions. In some aspects, the disclosureprovides compositions, that include such modified small-moleculecytotoxins and a protein, such as albumin or albumin mimetics. Further,the disclosure provides various uses of these compounds andcompositions.

DESCRIPTION OF RELATED ART

Cancer refers to a group of diseases characterized by the formation ofmalignant tumors or neoplasms, which involve abnormal cell growth andhave the potential to invade adjacent tissue and spread to other partsof the body. There are more than 14 million new diagnoses of cancerannually. Moreover, cancer accounts for more than 8 million deaths eachyear, which is about 15% of all deaths worldwide. In developedcountries, cancer accounts for an even higher percentage of deaths.

Therapies for cancer have improved significantly over the years. Inparticular, an increasing number of cytotoxic agents have beendiscovered. These agents generally work by killing the cancer cells. Butcytotoxic agents can be harmful to normal cells as well. Therefore,subjects undergoing treatment with such agents often suffer certainside-effects from the treatment. In some cases, the side-effects posesuch a substantial risk that it may be necessary to administer verylimited quantities of cytotoxic agents. So, while there is a generaldesire to discover increasingly toxic chemotherapeutic agents, it isalso desirable to develop new means of directing those compoundsselectively to cancer cells and away from normal cells.

Various strategies have been used to assist in directingchemotherapeutic agents selectively to cancer cells. For example,certain compounds rely on passive targeting, where the compound isselectively directed toward cancer cells (e.g., in a solid tumor) as aresult of the fact that cancer cells tend to divide more rapidly thanother cells and therefore have a higher appetite for certain biologicalbuilding blocks. For example, gemcitabine, a commonly used cytotoxin,contains a sugar-like moiety as well as a toxic payload (a5-fluorouracil moiety). Because cancer cells may have a higher need forsugar than other cells, gemcitabine is passively drawn to rapidlydividing cancer cells because it looks like a sugar molecule. Once atthe cancer cell, gemcitabine can release its toxic payload. In someother cases, the targeting may be active, where the cytotoxic agentincludes a moiety that binds selectively to a protein that isoverexpressed in certain cancer cells. For example, pemetrexed includesa moiety that mimics folic acid, and thereby allows the drug to activelytarget cancer cells that overexpress folic acid receptors.

Such passive and active targeting has allowed for the development ofincreasingly toxic cytotoxic agents that have reduced side-effects withrespect to earlier-generation cytotoxins. Even so, progress has beenslow, and it is often impractical to build certain features intocytotoxic agents that allow them to target cancer cells selectively andalso be formulated in a way that is suitable for delivery. Therefore,there is a continuing need to discover new ways of selectively directingcytotoxic agents to cancer cells.

SUMMARY

The present disclosure provides compounds and compositions that candeliver increasingly toxic quantities of a cytotoxin to cancer cells(e.g., in a solid tumor) with reduced side-effects with respect to othernon-cancerous cells. In some embodiments, the compounds are prodrugs ofsmall-molecule cytotoxins, such that the prodrug permits improveddelivery of the cytotoxin to a solid tumor in a mammal. The disclosurealso provides methods and uses of those compounds and compositions forthe treatment of cancer.

In a first aspect, the disclosure provides compounds of formula (I):A¹-X¹—X²-A²  (I)wherein: A¹ is an organic group, or is a hydrophilic group, or ahydrogen atom; A² is a cytotoxic drug moiety, which has a molecularweight of no more than 1600 Da; X¹ is a hydrophobic group; and X² is adirect bond, an organic group, or a heteroatom group selected from thegroup consisting of —O—, —S—, —S(═O), —S(═O)₂—, —S—S—, —N═, ═N—, —N(H)—,—N═N—N(H)—, —N(H)—N═N—, —N(OH)—, or —N(═O)—. In some embodiments, A¹ isa hydrophilic group, such as a carboxylic acid group (—COOH) orpharmaceutically acceptable salts thereof. In some embodiments, thecytotoxic drug moiety is a paclitaxel moiety, a doxorubicin moiety, or apemetrexed moiety. In some embodiments, the hydrophobic group is aC₁₂₋₂₂ hydrocarbylene group, which is optionally substituted. In someembodiments, X² is an organic group, such as a carbonyl group, i.e.,—C(═O)—.

In a second aspect, the disclosure provides a compound of the followingformula:

or a pharmaceutically acceptable salt thereof.

In a third aspect, the disclosure provides compositions (e.g.,pharmaceutical compositions) that include: a compound of any embodimentsof the foregoing aspects; and a protein. In some embodiments, theprotein is an albumin or an albumin mimetic.

In a fourth aspect, the disclosure provides compositions (e.g.,pharmaceutical compositions) that include: a compound of any embodimentsof the first or second aspects; a protein, wherein the protein is analbumin or an albumin mimetic; and a carrier, which includes water;wherein the compound and the protein are non-covalently associated witheach other; and wherein the compound and the protein are solvated by thecarrier.

In a fifth aspect, the disclosure provides methods of treating cancer,which include administering to a subject a compound or composition ofany embodiments of any of the foregoing aspects.

In a sixth aspect, the disclosure provides methods of inducing apoptosisin a cancer cell, which include contacting the cancer cell with acompound or composition of any embodiments of any of the first throughthe fourth aspects.

In a seventh aspect, the disclosure provides methods for inhibitinggrowth of a cancerous tumor, which includes contacting the canceroustumor with a compound of any embodiments of the first or second aspects.

In an eighth aspect, the disclosure provides uses of a compound orcomposition of any embodiments of any of the first through the fourthaspects as a medicament.

In a ninth aspect, the disclosure provides uses of a compound orcomposition of any embodiments of any of the first through the fourthaspects for treating cancer.

In a tenth aspect, the disclosure provides uses of a compound orcomposition of any embodiments of any of the first through the fourthaspects in the manufacture of a medicament.

In an eleventh aspect, the disclosure provides uses of a compound orcomposition of any embodiments of any of the first through the fourthaspects in the manufacture of a medicament for treating cancer.

In a twelfth aspect, the disclosure provides methods of making compoundsof the first and second aspects and compositions of the third and fourthaspects.

Further aspects and embodiments are provided in the drawings, thedetailed description, the claims, and the abstract.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings are provided for purposes of illustrating variousembodiments of the compounds, compositions, methods, and uses disclosedherein. The drawings are provided for illustrative purposes only, andare not intended to describe any preferred compounds or compositions orany preferred methods or uses, or to serve as a source of anylimitations on the scope of the claimed inventions.

FIG. 1 shows a non-limiting example of a compound of formula (I), wherethe compound is paclitaxel, which is modified to include an ester ofoctadecanedioic acid.

FIG. 2 shows micrograph of a cryo-TEM analysis of the PTX-FA/HSAformulation.

DETAILED DESCRIPTION

The following description recites various aspects and embodiments of theinventions disclosed herein. No particular embodiment is intended todefine the scope of the invention. Rather, the embodiments providenon-limiting examples of various compositions, and methods that areincluded within the scope of the claimed inventions. The description isto be read from the perspective of one of ordinary skill in the art.Therefore, information that is well known to the ordinarily skilledartisan is not necessarily included.

Definitions

The following terms and phrases have the meanings indicated below,unless otherwise provided herein. This disclosure may employ other termsand phrases not expressly defined herein. Such other terms and phrasesshall have the meanings that they would possess within the context ofthis disclosure to those of ordinary skill in the art. In someinstances, a term or phrase may be defined in the singular or plural. Insuch instances, it is understood that any term in the singular mayinclude its plural counterpart and vice versa, unless expresslyindicated to the contrary.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to “a substituent” encompasses a single substituent as well astwo or more substituents, and the like.

As used herein, “for example,” “for instance,” “such as,” or “including”are meant to introduce examples that further clarify more generalsubject matter. Unless otherwise expressly indicated, such examples areprovided only as an aid for understanding embodiments illustrated in thepresent disclosure, and are not meant to be limiting in any fashion. Nordo these phrases indicate any kind of preference for the disclosedembodiment.

As used herein, “hydrocarbon” refers to an organic group composed ofcarbon and hydrogen, which can be saturated or unsaturated, and caninclude aromatic groups. The term “hydrocarbyl” refers to a monovalentor polyvalent (e.g., divalent or higher) hydrocarbon moiety. In somecases, a divalent hydrocarbyl group is referred to as a “hydrocarbylene”group.

As used herein, “alkyl” refers to a straight or branched chain saturatedhydrocarbon having 1 to 30 carbon atoms, which may be optionallysubstituted, as herein further described, with multiple degrees ofsubstitution being allowed. Examples of “alkyl,” as used herein,include, but are not limited to, methyl, ethyl, n-propyl, isopropyl,isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl,neopentyl, n-hexyl, and 2-ethylhexyl. In some instances, the “alkyl”group can be divalent, in which case, the group can alternatively bereferred to as an “alkylene” group. Also, in some instances, one or moreof the carbon atoms in the alkyl or alkylene group can be replaced by aheteroatom (e.g., selected from nitrogen, oxygen, or sulfur, includingN-oxides, sulfur oxides, sulfur dioxides, and carbonyl groups, wherefeasible), and is referred to as a “heteroalkyl” or “heteroalkylene”group, respectively. Non-limiting examples include “oxyalkyl” or“oxyalkylene” groups, which refer to groups where a carbon atom in thealkyl or alkylene group is replaced by oxygen. Non-limiting examples ofoxyalkyl or oxyalkylene groups include alkyl or alkylene chains thatcontain a carbonyl group, and also alkoxylates, polyalkylene oxides, andthe like.

The number of carbon atoms in any group or compound can be representedby the terms. Thus, “C_(z)” refers to a group of compound having zcarbon atoms, and “C_(x-y)”, refers to a group or compound containingfrom x to y, inclusive, carbon atoms. For example, “C₁₋₆ alkyl”represents an alkyl group having from 1 to 6 carbon atoms and, forexample, includes, but is not limited to, methyl, ethyl, n-propyl,isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl,n-pentyl, neopentyl, and n-hexyl. The same logic applies to other typesof functional groups, defined below.

As used herein, “alkenyl” refers to a straight or branched chainnon-aromatic hydrocarbon having 2 to 30 carbon atoms and having one ormore carbon-carbon double bonds, which may be optionally substituted, asherein further described, with multiple degrees of substitution beingallowed. Examples of “alkenyl,” as used herein, include, but are notlimited to, ethenyl, 2-propenyl, 2-butenyl, and 3-butenyl. In someinstances, the “alkenyl” group can be divalent, in which case the groupcan alternatively be referred to as an “alkenylene” group. Also, in someinstances, one or more of the carbon atoms in the alkenyl or alkenylenegroup can be replaced by a heteroatom (e.g., selected from nitrogen,oxygen, or sulfur, including N-oxides, sulfur oxides, sulfur dioxides,and carbonyl groups, where feasible), and is referred to as a“heteroalkenyl” or “heteroalkenylene” group, respectively.

As used herein, “cycloalkyl” refers to an aliphatic saturated orunsaturated hydrocarbon ring system having 3 to 20 carbon atoms, whichmay be optionally substituted, as herein further described, withmultiple degrees of substitution being allowed. In some embodiments, theterm refers only to saturated hydrocarbon ring systems, substituted asherein further described. Examples of “cycloalkyl,” as used herein,include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, adamantyl, and thelike. In some instances, the “cycloalkyl” group can be divalent, inwhich case the group can alternatively be referred to as a“cycloalkylene” group. Cycloalkyl and cycloalkylene groups can also bereferred to herein as “carbocyclic rings.” Also, in some instances, oneor more of the carbon atoms in the cycloalkyl or cycloalkylene group canbe replaced by a heteroatom (e.g., selected independently from nitrogen,oxygen, silicon, or sulfur, including N-oxides, sulfur oxides, andsulfur dioxides, where feasible), and is referred to as a “heterocyclyl”or “heterocyclylene” group, respectively. The term “heterocyclic ring”can also be used interchangeably with either of these terms. In someembodiments, the cycloalkyl and heterocyclyl groups are fully saturated.In some other embodiments, the cycloalkyl and heterocyclyl groups cancontain one or more carbon-carbon double bonds.

As used herein, “halogen,” “halogen atom,” or “halo” refer to afluorine, chlorine, bromine, or iodine atom. In some embodiments, theterms refer to a fluorine or chlorine atom.

As used herein, the terms “organic group,” “organic moiety,” or “organicresidue” refer to a monovalent or polyvalent functional group having atleast one carbon atom, which optionally contains one or more additionalatoms selected from the group consisting of hydrogen atoms, halogenatoms, nitrogen atoms, oxygen atoms, phosphorus atoms, and sulfur atoms,and which does not include covalently bound metal or semi-metal atoms.In some embodiments, these terms can include metal salts of organicgroups, such as alkali metal or alkaline earth metal salts of organicanions.

As used herein, the term “pharmacophore” refers to a type of organicfunctional group. Standard pharmacophores are hydrophobicpharmacophores, hydrogen-bond donating pharmacophores, hydrogen-bondaccepting pharmacophores, positive ionizable pharmacophores, andnegative ionizable pharmacophores. The classification of organicfunctional groups within a compound is carried out according to standardclassification systems known in the art.

As used herein, the terms “hydrophobic group,” “hydrophobic moiety,” or“hydrophobic residue” refer to an organic group that consistsessentially of hydrophobic pharmacophores. In some embodiments, theterms refer to an organic group that consists of hydrophobicpharmacophores.

As used herein, the terms “hydrophilic group,” “hydrophilic moiety,” or“hydrophilic residue” refer to an organic group that comprises onepharmacophores selected from the group consisting of hydrogen bonddonors, hydrogen bond acceptors, negative ionizable groups, or positiveionizable groups. In some embodiments, the terms refer to an organicgroup that consist essentially of pharmacophores selected from the groupconsisting of hydrogen bond donors, hydrogen bond acceptors, negativeionizable groups, or positive ionizable groups.

As used herein, the term “drug moiety” refers to a drug compound, or apharmaceutically acceptable salt thereof, where an atom or a group ofatoms is absent, thereby creating a monovalent or polyvalent moiety. Insome embodiments, for example, a hydrogen atom is absent, therebycreating a monovalent moiety. In some other embodiments, a functionalgroup, such as an —OH moiety, an —NH₂ moiety, or a —COOH, moiety isabsent. In some embodiments, the drug moiety is a “cytotoxic drugmoiety,” which refers to a drug moiety (as defined above) of a cytotoxicdrug compound. One non-limiting example of such a “drug moiety,” (inthis case, a “paclitaxel moiety”) is the moiety of the followingformula:

where a hydrogen atom is absent to create a monovalent moiety that,within a compound, bonds to the rest of the molecule through theremaining oxygen atom. Note that the term “drug moiety” is not limitedto any particular procedure for making such compounds.

Various methods of drawing chemical structures are used herein. In someinstances, the bond line-structure method is used to depict chemicalcompounds or moieties. In the line-structure method, the lines representchemical bonds, and the carbon atoms are not explicitly shown (but areimplied by the intersection of the lines). The hydrogen atoms are alsonot explicitly shown, except in instances where they are attached toheteroatoms. Heteroatoms, however, are explicitly shown. Thus, usingthat methodology, the structures shown below are for 2-methylpropane,1-methoxypropane, and 1-propanol:

In that methodology, aromatic rings are typically represented merely byone of the contributing resonance structures. Thus, the followingstructures are for benzene, pyridine, and pyrrole:

As used herein, a “protein binding moiety” is a moiety that bindsnon-covalently to one or more sites on a protein with a binding constant(K_(b)) of at least 100 M⁻¹ in water at 25° C.

As used herein, “amino acid” refers to a compound having the structureH₂N—R^(x)—COOH, where R^(x) is an organic group, and where the NH₂ mayoptionally combine with R^(x) (e.g., as in the case of proline). Theterm includes any known amino acids, including, but not limited to,alpha amino acids, beta amino acids, gamma amino acids, delta aminoacids, and the like. In some embodiments, the term can refer to alphaamino acids.

As used herein, “hydroxy acid” refers to a compound having the structureHO—R^(y)—COOH, where R^(y) is an organic group. Non-limiting examplesinclude glycolic acid, lactic acid, and caprolactone.

As used herein, “alkanol amine” refers to a compound having thestructure HO—R^(z)—NH₂, where R^(z) is an optionally substitutedalkylene group. Non-limiting examples include ethanol amine.

As used herein, “administer” or “administering” means to introduce, suchas to introduce to a subject a compound or composition. The term is notlimited to any specific mode of delivery, and can include, for example,subcutaneous delivery, intravenous delivery, intramuscular delivery,intracisternal delivery, delivery by infusion techniques, transdermaldelivery, oral delivery, nasal delivery, and rectal delivery.Furthermore, depending on the mode of delivery, the administering can becarried out by various individuals, including, for example, ahealth-care professional (e.g., physician, nurse, etc.), a pharmacist,or the subject (i.e., self-administration).

As used herein, “treat” or “treating” or “treatment” can refer to one ormore of: delaying the progress of a disease, disorder, or condition;controlling a disease, disorder, or condition; ameliorating one or moresymptoms characteristic of a disease, disorder, or condition; ordelaying the recurrence of a disease, disorder, or condition, orcharacteristic symptoms thereof, depending on the nature of the disease,disorder, or condition and its characteristic symptoms.

As used herein, “subject” refers to any mammal such as, but not limitedto, humans, horses, cows, sheep, pigs, mice, rats, dogs, cats, andprimates such as chimpanzees, gorillas, and rhesus monkeys. In someembodiments, the “subject” is a human. In some such embodiments, the“subject” is a human who exhibits one or more symptoms characteristic ofa disease, disorder, or condition. The term “subject” does not requireone to have any particular status with respect to a hospital, clinic, orresearch facility (e.g., as an admitted patient, a study participant, orthe like).

As used herein, the term “compound” includes free acids, free bases, andsalts thereof

As used herein, the term “pharmaceutical composition” is used to denotea composition that may be administered to a mammalian host, e.g.,orally, topically, parenterally, by inhalation spray, or rectally, inunit dosage formulations containing conventional non-toxic carriers,diluents, adjuvants, vehicles and the like. The term “parenteral” asused herein, includes subcutaneous injections, intravenous,intramuscular, intracisternal injection, or by infusion techniques.

Also included within the scope of the disclosure are the individualenantiomers of the compounds represented by Formula (I) orpharmaceutically acceptable salts thereof, as well as any wholly orpartially racemic mixtures thereof. The disclosure also covers theindividual enantiomers of the compounds represented by Formula (I) orpharmaceutically acceptable salts thereof, as well as mixtures withdiastereoisomers thereof in which one or more stereocenters areinverted. Unless otherwise stated, structures depicted herein are alsomeant to include compounds which differ only in the presence of one ormore isotopically enriched atoms. For example, compounds having thepresent structure, except for the replacement of a hydrogen atom by adeuterium or tritium, or the replacement of a carbon atom by a ¹³C- or¹⁴C-enriched carbon are within the scope of the disclosure.

As used herein, “mix” or “mixed” or “mixture” refers broadly to anycombining of two or more compositions. The two or more compositions neednot have the same physical state; thus, solids can be “mixed” withliquids, e.g., to form a slurry, suspension, or solution. Further, theseterms do not require any degree of homogeneity or uniformity ofcomposition. This, such “mixtures” can be homogeneous or heterogeneous,or can be uniform or non-uniform. Further, the terms do not require theuse of any particular equipment to carry out the mixing, such as anindustrial mixer.

As used herein, “optionally” means that the subsequently describedevent(s) may or may not occur. In some embodiments, the optional eventdoes not occur. In some other embodiments, the optional event does occurone or more times.

As used herein, “substituted” refers to substitution of one or morehydrogen atoms of the designated moiety with the named substituent orsubstituents, multiple degrees of substitution being allowed unlessotherwise stated, provided that the substitution results in a stable orchemically feasible compound. A stable compound or chemically feasiblecompound is one in which the chemical structure is not substantiallyaltered when kept at a temperature from about −80° C. to about +40° C.,in the absence of moisture or other chemically reactive conditions, forat least a week. As used herein, the phrases “substituted with one ormore . . . ” or “substituted one or more times . . . ” refer to a numberof substituents that equals from one to the maximum number ofsubstituents possible based on the number of available bonding sites,provided that the above conditions of stability and chemical feasibilityare met.

As used herein, “comprise” or “comprises” or “comprising” or “comprisedof” refer to groups that are open, meaning that the group can includeadditional members in addition to those expressly recited. For example,the phrase, “comprises A” means that A must be present, but that othermembers can be present too. The terms “include,” “have,” and “composedof” and their grammatical variants have the same meaning. In contrast,“consist of” or “consists of” or “consisting of” refer to groups thatare closed. For example, the phrase “consists of A” means that A andonly A is present. As used herein, the phrases “consist essentially of,”“consists essentially of,” and “consisting essentially of” refer togroups that are open, but which only includes additional unnamed membersthat would not materially affect the basic characteristics of theclaimed subject matter.

As used herein, “or” is to be given its broadest reasonableinterpretation, and is not to be limited to an either/or construction.Thus, the phrase “comprising A or B” means that A can be present and notB, or that B is present and not A, or that A and B are both present.Further, if A, for example, defines a class that can have multiplemembers, e.g., A₁ and A₂, then one or more members of the class can bepresent concurrently.

As used herein, the various functional groups represented will beunderstood to have a point of attachment at the functional group havingthe hyphen or dash (—) or a dash used in combination with an asterisk(*). In other words, in the case of —CH₂CH₂CH₃ or *—CH₂CH₂CH₃, it willbe understood that the point of attachment is the CH₂ group at the farleft. If a group is recited without an asterisk or a dash, then theattachment point is indicated by the plain and ordinary meaning of therecited group.

As used herein, multi-atom bivalent species are to be read from left toright. For example, if the specification or claims recite A-D-E and D isdefined as —OC(O)—, the resulting group with D replaced is: A-OC(O)-Eand not A-C(O)O-E.

Other terms are defined in other portions of this description, eventhough not included in this subsection.

Modified Cytotoxins

In at least one aspect, the disclosure provides compounds of formula(I):A¹-X¹—X²-A²  (I)wherein: A¹ is a hydrophilic group or a hydrogen atom, or is an organicgroup; A² is a cytotoxic drug moiety; X¹ is a hydrophobic group; and X²is a direct bond, an organic group, or a group selected from the groupconsisting of —O—, —S—, —S(═O)—, —S(═O)₂—, —S—S—, —N═, ═N—, —N(H)—,—N═N—N(H)—, —N(H)—N═N—, —N(OH)—, or —N(═O)—.

In some embodiments, A¹ is an organic group. A¹ can contain any suitablenumber of carbon atoms. In some embodiments, for example, A¹ containsfrom 1 to 100 carbon atoms, or from 1 to 50 carbon atoms, or from 1 to25 carbon atoms, or from 1 to 10 carbon atoms, or from 1 to 6 carbonatoms. A¹ can also contain one or more heteroatoms, such as nitrogen,oxygen, sulfur, or phosphorus.

In some embodiments according to any of the foregoing embodiments, A¹ isa hydrophilic group or moiety. Non-limiting examples of a hydrophilicgroup include, but are not limited to, a carboxylic acid moiety, anester moiety, an amide moiety, a urea moiety, an amine moiety, an ethermoiety, an alcohol moiety, a thioether moiety, a thiol moiety, a ketonemoiety, an aldehyde moiety, a sulfate moiety, a thiosulfate moiety, asulfite moiety, a thiosulfite moiety, a phosphate moiety, a phosphonatemoiety, a phosphinate moiety, a phosphite moiety, a borate moiety, or aboronate moiety.

In some embodiments of any of the aforementioned embodiments, A¹ isselected from the group consisting of a carboxylic acid group (—COOH), acarboxylate anion (—COO⁻), or a carboxylate ester (—COOR^(a), whereR^(a) is an organic group such as an alkyl or alkoxylate group). In somesuch embodiments, A¹ is a carboxylic acid group. In some suchembodiments, A¹ is a carboxylate ester group.

In some other embodiments of any of the aforementioned embodiments, A¹is a hydrogen atom. In some other embodiments of any of theaforementioned embodiments, A¹ is a hydroxyl (—OH) group.

In any of the aforementioned embodiments, X¹ can be a hydrophobic grouphaving any suitable number of carbon atoms. In some embodiments, forexample, X¹ contains from 1 to 100 carbon atoms, or from 1 to 50 carbonatoms, or from 1 to 25 carbon atoms.

In some embodiments of any of the aforementioned embodiments, X¹ isC₈₋₃₀ hydrocarbylene, which is optionally substituted. In some furtherembodiments, X¹ is C₁₂₋₂₂ hydrocarbylene, which is optionallysubstituted. In some further embodiments, X¹ is C₁₂₋₂₂ alkylene. In somefurther embodiments, X¹ is —(CH₂)₁₂—, —(CH₂)₁₄—, —(CH₂)₁₆—, —(CH₂)₁₈—,—(CH₂)₂₀—, or —(CH₂)₂₂—. In some other embodiments, X¹ is —(CH₂)₁₆—. Insome further embodiments, X¹ is C₁₂₋₂₂ alkenylene. In some further suchembodiments, X¹ is —(CH₂)₇—CH═CH—(CH₂)₇—.

In some further embodiments of any of the aforementioned embodiments, X¹is C₁₂₋₂₂ hydrocarbylene, which is optionally substituted. In some suchembodiments, X¹ is C₁₂₋₂₂ hydrocarbylene. In some further suchembodiments, X¹ is C₁₄₋₂₂ hydrocarbylene. In some further suchembodiments, X¹ is C₁₆₋₂₂ hydrocarbylene. In some embodiments of any ofthe aforementioned embodiments, X¹ is C₁₂₋₂₂ hydrocarbylene, wherein A¹and X² (or, if X² is a direct bond, A²) are separated from each other byat least 6, or by at least 8, or by at least 10, or by at least 12, orby at least 14, carbon atoms. In some further such embodiments, X¹ isC₁₄₋₂₂ hydrocarbylene, wherein A¹ and X² (or, if X² is a direct bond,A²) are separated from each other by at least 6, or by at least 8, or byat least 10, or by at least 12, or by at least 14, carbon atoms. In somefurther such embodiments, X¹ is C₁₆₋₂₂ hydrocarbylene, wherein A¹ and X²(or, if X² is a direct bond, A²) are separated from each other by atleast 6, or by at least 8, or by at least 10, or by at least 12, or byat least 14, carbon atoms. In some further embodiments of any of theaforementioned embodiments, X¹ is C₁₂₋₂₂ straight-chain alkylene, orC₁₄₋₂₂ straight-chain alkylene, or C₁₆₋₂₂ straight-chain alkylene. Insome further embodiments of any of the aforementioned embodiments, X¹ isC₁₂₋₂₂ straight-chain alkenylene, or C₁₄₋₂₂ straight-chain alkenylene,or C₁₆₋₂₂ straight-chain alkenylene.

In some embodiments of any of the aforementioned embodiments, X² is adirect bond. In some other embodiments of any of the aforementionedembodiments, X² is an organic group. In some embodiments, X² is ahydrophilic group. In some embodiments, X² is a heteroalkylene group.

In any of the aforementioned embodiments where X² is an organic group,X² can contain any suitable number of carbon atoms. In some embodiments,for example, X² contains from 1 to 100 carbon atoms, or from 1 to 50carbon atoms, or from 1 to 25 carbon atoms, or from 1 to 10 carbonatoms, or from 1 to 6 carbon atoms.

In any of the aforementioned embodiments where X² is a heteroalkylenegroup, X² can contain any suitable number of carbon atoms. In someembodiments, for example, X² contains from 1 to 100 carbon atoms, orfrom 1 to 50 carbon atoms, or from 1 to 25 carbon atoms, or from 1 to 10carbon atoms, or from 1 to 6 carbon atoms.

In some of the aforementioned embodiments, X² can contain certaingroups. Some non-limiting examples of such groups that X² can containare polyalkylene oxide groups, such as polyethylene glycol (PEG) andvarious polypeptide chains.

In some embodiments, X² is an organic group selected from the groupconsisting of —C(═O)—, —C(H)≡C(H)—, —C(═O)—O—, —O—C(═O)—, —C(═O)—NH—,—NH—C(═O)—, —NH—C(═O)—O—, —O—(C═O)—NH—, —O—C(═O)—O—, —C(═N—NH₂)—,—C(═N—R^(b))— (where R^(b) is a hydrogen atom or an alkyl group),—C(═N—OH)—, —NH—C(═O)—NH—, —NH—C(═S)—NH—, —NH—C(═S)—O—, —O—C(═S)—NH—,—NH—C(═O)—S—, —S—C(═O)—NH—, —NH—C(═S)—S—, —S—C(═S)—NH—, and the cyclicstructures shown below:

where R^(c), R^(d), and R^(e) are, independently at each occurrence, ahydrogen atom or C₁₋₁₀ alkyl. In some further embodiments, X² is—C(═O)—.

In some embodiments, X² is a group selected from the group consisting of—O—, —S—, —S(═O)—, —S(═O)₂—, —S—S—, —N═, ═N—, —N(H)—, —N═N—N(H)—,—N(H)—N═N—, —N(OH)—, and —N(O)—.

In some embodiments, X² comprises one or more moieties selected from thegroup consisting of: —C(═O)—, —O—C(═O)—, —NH—C(═O)—, one or moremoieties formed from a alkylene glycols, one or more units formed fromalkanol amines, one or more units formed from amino acids, and one ormore units formed from hydroxyl acids. Thus, in some embodiments, X²comprises one or more moieties formed from alkylene glycols, such as ashort poly(ethylene glycol) chain having 1 to 25 ethylene glycol units.In some embodiments, X² comprises one or more moieties formed from aminoacids, such as an oligopeptide chain having 1 to 25 amino acid units. Insome embodiments, X² comprises one or more moieties formed from hydroxyacids, such as moieties formed from glycolic acid, lactic acid, orcaprolactone. In some embodiments, X² comprises a combination of apoly(ethylene glycol) chain having 1 to 25 ethylene glycol units and anoligopeptide having 1 to 25 amino acid units, and optionally one or moreunits formed from hydroxy acids.

In any of the above embodiments, the selection of X² will depend on thetype of functional group through which it is linked to the cytotoxicdrug moiety, so as to avoid making compounds that are chemicallyunstable or impossible. The skilled artisan will be able to selectcombinations of X² and A² that result in chemically stable compounds,which are compounds in which the chemical structure is not substantiallyaltered when kept at a temperature from about −80° C. to about +40° C.,in the absence of moisture or other chemically reactive conditions, forat least a week.

In the above embodiments, A² can be any suitable cytotoxic drug moiety.In some embodiments, the cytotoxic drug moiety is a small-molecule drugmoiety, such as a cytotoxic drug moiety having a molecular weight of orno more than 1600 Da, or no more than 1500 Da, or no more than 1400 Da,or no more than 1300 Da, no more than 1200 Da, or no more than 1100 Da,or no more than 1000 Da, or no more than 900 Da. Such drug moieties canbe organic moieties, or can also be moieties that contain inorganicatoms (e.g., platinum). In some embodiments, however, the cytotoxic drugmoiety is an organic moiety.

In some embodiments of any of the aforementioned embodiments, thecytotoxic drug moiety is a moiety selected from the group consisting of:a paclitaxel moiety, an etoposide moiety, a gemcitabine moiety, acyclophosphamide moiety, a chlorambucil moiety, a doxorubicin moiety, adaunorubicin moiety, a 5-fluorouracil moiety, a dactinomycin moiety, anamifostine moiety, a fludarabine moiety, a topotecan moiety, anifosfamide moiety, a vincristine moiety, a carboplatin moiety, avinblastine moiety, an imatinib moiety, a lenalidomide moiety, apemetrexed moiety, an abiraterone moiety, an erlotinib moiety, abortezomib moiety, an oxaliplatin moiety, a methotrexate moiety, acarfilzomib moiety, a crizotinib moiety, a vismodegib moiety, aponatinib moiety, a tivozanib moiety, a carbozantinib moiety, anepirubicin moiety, a docetaxel moiety, a cisplatin moiety, an eribulunmoiety, an ixabepilone moiety, a vinorelbine moiety, an everolimusmoiety, a mytomycin C moiety, a sunitinib moiety, an irinotecan moiety,a leicovorim moiety, a tretinoin moiety, an allopurinol moiety, anasparaginase moiety, a bendamustine moiety, a bleomycin moiety, afolinic acid moiety, a capecitabine moiety, a cytarabine moiety, adacarbazine moiety, a filgrastim moiety, a hydroxycarbamide moiety, amercaptopurine moiety, a mesna moiety, a procarbazine moiety, athioguanine moiety, and pharmaceutically acceptable salts of any of theforegoing. In some further such embodiments, the cytotoxic drug moietyis a moiety selected from the group consisting of: a paclitaxel moiety,an etoposide moiety, a gemcitabine moiety, a cyclophosphamide moiety, achlorambucil moiety, a doxorubicin moiety, a daunorubicin moiety, a5-fluorouracil moiety, a dactinomycin moiety, an amifostine moiety, afludarabine moiety, a topotecan moiety, an ifosfamide moiety, avincristine moiety, a vinblastine moiety, an imatinib moiety, alenalidomide moiety, a pemetrexed moiety, an abiraterone moiety, anerlotinib moiety, a bortezomib moiety, a methotrexate moiety, acarfilzomib moiety, a crizotinib moiety, a vismodegib moiety, aponatinib moiety, a tivozanib moiety, a carbozantinib moiety, anepirubicin moiety, a docetaxel moiety, an eribulun moiety, anixabepilone moiety, a vinorelbine moiety, an everolimus moiety, amytomycin C moiety, a sunitinib moiety, an irinotecan moiety, aleicovorim moiety, and pharmaceutically acceptable salts of any of theforegoing. In some further such embodiments, the cytotoxic drug moietyis selected from the group consisting of: a paclitaxel moiety, agemcitabine moiety, a doxorubicin moiety, a 5-fluorouracil moiety, amethotrexate moiety, and a pemetrexed moiety. In some further suchembodiments, the cytotoxic drug moiety is a paclitaxel moiety. In somefurther such embodiments, the cytotoxic drug moiety is a gemcitabinemoiety. In some further such embodiments, the cytotoxic drug moiety is a5-fluorouracil moiety. In some further such embodiments, the cytotoxicdrug moiety is a pemetrexed moiety.

In the aforementioned embodiments, the named moieties can have anysuitable chemical form. In some embodiments of any of the aforementionedembodiments, the cytotoxic drug moieties are moieties where a hydrogenatom is absent from the named drug compound, or a pharmaceuticallyacceptable salt thereof. As a non-limiting example, such a “paclitaxelmoiety” would include the moiety of the following formula:

In some instances, racemization may occur at the point of attachment ofthe moiety. Thus, another non-limiting example would include the moietyof the following formula:

The structures below show various non-limiting examples of compoundsthat are included within the scope of compounds of formula (I), asdefined above. Note that, at each occurrence, G is independently ahydrogen atom or —X²—X¹-A¹ (according to any of the aforementionedembodiments), wherein, for each compound, at least one G is not ahydrogen atom. In some embodiments, for each compound, exactly one G is—X²—X¹-A¹ (according to any of the aforementioned embodiments).

In the above structures, when G is —X²—X¹-A¹, it can be —X²—X¹-Aaccording to any of the previously recited embodiments, so long as thosecombinations result in stable chemical structures that would be suitablefor pharmaceutical use. In some such embodiments, however, —X²—X¹-A¹ is—C(═O)—(CH₂)₁₀—CH₃, —C(═O)—(CH₂)₁₂—CH₃, —C(═O)—(CH₂)₁₄—CH₃, or—C(═O)—(CH₂)₁₆—CH₃. In some other such embodiments, —X²—X¹-A¹ is—C(═O)—(CH₂)₁₀—C(═O)—OH, —C(═O)—(CH₂)₁₂—C(═O)—OH,—C(═O)—(CH₂)₁₄—C(═O)—OH, or —C(═O)—(CH₂)₁₆—C(═O)—OH.

The selection of —X²—X¹-A¹ can depend on the nature of the connection tothe drug moiety.

For example, in embodiments where the —X²—X¹-A¹ connects to an oxygenatom or an NH group on the drug moiety, as is the case for entries HA1,HA2, HA9, HA12, HA14, HA15, HA16, HA19, HA20, HA21, HA22, HA23, and HA24in Table 1, then —X²—X¹-A¹ is selected from the group consisting of:—C(═O)—(CH₂)_(n1)—C(═O)—OH; —C(═O)—(CH₂)_(n1)—C(═O)—OCH₃;—C(═O)—(CH₂)_(n1)—CH₃; —C(═O)—(C₁₋₆alkylene)-C(═O)—O—(CH₂)_(n2)—C(═O)—OH; —C(═O)—(C₁₋₆alkylene)-NH—C(═O)—(CH₂)_(n1)—C(═O)—OH; —C(═O)—(C₁₋₆alkylene)-C(═O)—O—[(CH₂)₂—O—]_(n3)(CH₂)_(n2)—C(═O)—OH;—C(═O)—O—(CH₂)_(n2)—C(═O)—OH; and —C(═O)—NH—(CH₂)_(n2)—C(═O)—OH; whereinn1 is an integer 12 to 24, n2 is an integer from 13 to 25, and n3 is aninteger from 1 to 25. In some further such embodiments, —X²—X¹-A¹ isselected from the group consisting of: —C(═O)—(CH₂)_(n1)—C(═O)—OH;—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃; —C(═O)—(C₁₋₆alkylene)-C(═O)—O—(CH₂)_(n2)—C(═O)—OH; —C(═O)—(C₁₋₆alkylene)-NH—C(═O)—(CH₂)_(n1)—C(═O)—OH; —C(═O)—(C₁₋₆alkylene)-C(═O)—O—[(CH₂)₂—O—]_(n3)(CH₂)_(n2)—C(═O)—OH;—C(═O)—O—(CH₂)_(n2)—C(═O)—OH; and —C(═O)—NH—(CH₂)_(n2)—C(═O)—OH. In somefurther such embodiments, —X²—X¹-A¹ is selected from the groupconsisting of: —C(═O)—(CH₂)_(n1)—C(═O)—OH; —C(═O)—O—(CH₂)_(n2)—C(═O)—OH;and —C(═O)—NH—(CH₂)_(n2)—C(═O)—OH. In some other embodiments, —X²—X¹-A¹is —C(═O)—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH, where n1 is aninteger from 12 to 24. In some embodiments of any of the aforementionedembodiments, n1 is an integer from 14 to 22, or from 16 to 20. In someembodiments of any of the aforementioned embodiments, n2 is an integerfrom 15 to 23, or from 17 to 21. In some embodiments of any of theaforementioned embodiments, n3 is an integer from 1 to 15, or from 1 to10, or from 1 to 6. In some such embodiments, —X²—X¹-A¹ is —C(═O)—(C₁₋₆alkylene)-C(═O)—O—(CH₂)_(n3)—OH, where n3 is an integer from 14 to 26,or an integer from 16 to 24, or an integer from 18 to 22.

In embodiments where the —X²—X¹-A¹ connects to an >N group on the drugmoiety, as is the case for entries HA3 and HA4 in Table 1, then—X²—X¹-A¹ is selected from the group consisting of:—CH₂—O—C(═O)—(CH₂)_(n1)—C(═O)—OH; —CH₂—O—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃;—CH₂—O—C(═O)—(CH₂)_(n1)—CH₃; —CH₂—O—C(═O)—(C₁₋₆alkylene)-C(═O)—O—(CH₂)_(n2)—C(═O)—OH; —CH₂—O—C(═O)—(C₁₋₆alkylene)-NH—C(═O)—(CH₂)_(n1)—C(═O)—OH; —CH₂—O—C(═O)—(C₁₋₆alkylene)-C(═O)—O—[(CH₂)₂—O—]_(n3)(CH₂)_(n2)—C(═O)—OH;—CH₂—O—C(═O)—O—(CH₂)_(n2)—C(═O)—OH; and—CH₂—O—C(═O)—NH—(CH₂)_(n2)—C(═O)—OH; wherein n1 is an integer 12 to 24,n2 is an integer from 13 to 25, and n3 is an integer from 1 to 25. Insome further such embodiments, —X²—X¹-A¹ is selected from the groupconsisting of: —CH₂—O—C(═O)—(CH₂)_(n1)—C(═O)—OH;—CH₂—O—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃; —CH₂—O—C(═O)—(C₁₋₆alkylene)-C(═O)—O—(CH₂)_(n2)—C(═O)—OH; —CH₂—O—C(═O)—(C₁₋₆alkylene)-NH—C(═O)—(CH₂)_(n1)—C(═O)—OH; —CH₂—O—C(═O)—(C₁₋₆alkylene)-C(═O)—O—[(CH₂)₂—O]_(n3)(CH₂)_(n2)—C(═O)—OH;—CH₂—O—C(═O)—O—(CH₂)_(n2)—C(═O)—OH; and —C(═O)—NH—(CH₂)_(n2)—C(═O)—OH.In some further such embodiments, —X²—X¹-A¹ is selected from the groupconsisting of: —CH₂—O—C(═O)—(CH₂)_(n1)—C(═O)—OH;—CH₂—O—C(═O)—O—(CH₂)_(n2)—C(═O)—OH; and—CH₂—O—C(═O)—NH—(CH₂)_(n2)—C(═O)—OH. In some embodiments of any of theaforementioned embodiments, n1 is an integer from 14 to 22, or from 16to 20. In some embodiments of any of the aforementioned embodiments, n2is an integer from 15 to 23, or from 17 to 21. In some embodiments ofany of the aforementioned embodiments, n3 is an integer from 1 to 15, orfrom 1 to 10, or from 1 to 6. In some such embodiments, —X²—X¹-A¹ is—CH₂—O—C(═O)—(C₁₋₆ alkylene)-C(═O)—O—(CH₂)_(n3)—OH, where n3 is aninteger from 14 to 26, or an integer from 16 to 24, or an integer from18 to 22.

In embodiments where the —X²—X¹-A¹ connects to a —C(═O) group on thedrug moiety, as is the case for entries HA5, HA6, HA7, HA8, HA11, andHA17 in Table 1, then —X²—X¹-A¹ is selected from the group consistingof: —O—(CH₂)_(n2)—C(═O)—OH; —NH—(CH₂)_(n2)-Q=O)—OH;—NH—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH;—O—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH;—NH—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃;—O—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃; —NH—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—CH₃; —O—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—CH₃; —NH—(C₁₋₆alkylene)-C(═O)—O—[(CH₂)₂—O—]_(n3)(CH₂)_(n2)—C(═O)—OH; and —O—(C₁₋₆alkylene)-C(═O)—O—[(CH₂)₂—O—]_(n3)(CH₂)_(n2)—C(═O)—OH; wherein n1 is aninteger 12 to 24, n2 is an integer from 13 to 25, and n3 is an integerfrom 1 to 25. In some further such embodiments, —X²—X¹-A¹ is selectedfrom the group consisting of: —O—(CH₂)_(n2)—C(═O)—OH;—NH—(CH₂)_(n2)—C(═O)—OH; —NH—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH;—O—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH;—NH—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃; and —O—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OCH₃. In some further suchembodiments, —X²—X¹-A¹ is selected from the group consisting of:—O—(CH₂)_(n2)—C(═O)—OH; —NH—(CH₂)_(n2)—(═O)—OH; —NH—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH; and—O—(C₁₋₆alkylene)-O—C(═O)—(CH₂)_(n1)—C(═O)—OH. In some embodiments ofany of the aforementioned embodiments, n1 is an integer from 14 to 22,or from 16 to 20. In some embodiments of any of the aforementionedembodiments, n2 is an integer from 15 to 23, or from 17 to 21. In someembodiments of any of the aforementioned embodiments, n3 is an integerfrom 1 to 15, or from 1 to 10, or from 1 to 6. In some such embodiments,—X²—X¹-A¹ is —O—(CH₂)_(n3)—OH, where n3 is an integer from 14 to 26, oran integer from 16 to 24, or an integer from 18 to 22.

In embodiments where the —X²—X¹-A¹ connects to a C═* group on the drugmoiety, as is the case for entries HA10, HA13, and HA18 in Table 1, then—X²—X¹-A¹ is selected from the group consisting of:═N—O—(CH₂)_(n2)—(C═O)—OH; ═N—NH—(CH₂)_(n2)—(C═O)—OH;═N—O—(CH₂)_(n2)—(C═O)—OCH₃; ═N—NH—(CH₂)_(n2)—(C═O)—OCH₃;═N—O—(CH₂)_(n2)—CH₃; ═N—NH—(CH₂)_(n2)—CH₃;═N—O—[(CH₂)₂O—]_(n3)(CH₂)_(n2)—C(═O)—OH; and═N—NH—[(CH₂)₂—O—]_(n3)(CH₂)_(n2)—C(═O)—OH; n2 is an integer from 13 to25, and n3 is an integer from 1 to 25. In some further such embodiments,—X²—X¹-A¹ is selected from the group consisting of:═N—O—(CH₂)_(n2)—(C═O)—OH; ═N—NH—(CH₂)_(n2)—(C═O)—OH;═N—O—(CH₂)_(n2)—(C═O)—OCH₃; and ═N—NH—(CH₂)_(n2)—(═O)—OCH₃. In somefurther such embodiments, —X²—X¹-A¹ is selected from the groupconsisting of: ═N—O—(CH₂)_(n2)—(═O)—OH and ═N—NH—(CH₂)_(n2)—C(═O)—OH. Insome embodiments of any of the aforementioned embodiments, n2 is aninteger from 15 to 23, or from 17 to 21. In some embodiments of any ofthe aforementioned embodiments, n3 is an integer from 1 to 15, or from 1to 10, or from 1 to 6. In some such embodiments, —X²—X¹-A¹ is selectedfrom the group consisting of: ═N—O—(CH₂)_(n3)—OH and═N—NH—(CH₂)_(n3)—OH, where n3 is an integer from 14 to 26, or an integerfrom 16 to 24, or an integer from 18 to 22.

The compounds described in any of the above embodiments can also existas pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salts” refers to salts of the compounds which are notbiologically or otherwise undesirable and are generally prepared byreacting the free base with a suitable organic or inorganic acid or byreacting the acid with a suitable organic or inorganic base.Representative salts include the following salts: acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, monopotassium maleate, mucate, napsylate, nitrate,N-methylglucamine, oxalate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium,stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide, trimethylammonium, and valerate. When an acidicsubstituent is present, such as —COOH, there can be formed the ammonium,morpholinium, sodium, potassium, barium, calcium salt, and the like, foruse as the dosage form. When a basic group is present, such as amino ora basic heteroaryl radical, such as pyridyl, there can be formed anacidic salt, such as hydrochloride, hydrobromide, phosphate, sulfate,trifluoroacetate, trichloroacetate, acetate, oxalate, maleate, pyruvate,malonate, succinate, citrate, tartarate, fumarate, mandelate, benzoate,cinnamate, methanesulfonate, ethanesulfonate, picrate, and the like.

The compounds above can be made by standard organic synthetic methods,such as those illustrated in: Wuts et al., Greene's Protective Groups inOrganic Synthesis (4th ed., 2006); Larock, Comprehensive OrganicTransformations (2nd ed., 1999); and Smith et al., March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure (6th ed., 2007).Specific non-limiting examples are shown below in the Examples.

The compounds of the foregoing embodiments, including theirpharmaceutically acceptable salts, are useful as cytotoxic agents orprodrugs thereof, and are therefore useful as compounds for thetreatment of cancer.

Table 3 (below) shows various examples of compounds that arecontemplated by the present disclosure. Table 3 refers to variouscombinations of an A²-moiety with a —X²—X¹-A¹, which together formcompounds of the present disclosure. Table 1 shows illustrative examplemoieties for the A²-moiety, wherein A² can be the moiety shown or canalso be a pharmaceutically acceptable salt thereof. Table 2 showsillustrative example moieties for —X²—X¹-A¹. Table 3 shows non-limitingillustrative combinations of the moieties from Tables 1 and 2, which cancome together to form compounds of the present disclosure. The compoundsdisclosed in Table 3 can be made by methods analogous to thoseillustrated in the Examples, and by common synthetic methods known tothose of ordinary skill in the art. Suitable methods of making suchcompounds are illustrated in: Wuts et al., Greene's Protective Groups inOrganic Synthesis (4th ed., 2006); Larock, Comprehensive OrganicTransformations (2nd ed., 1999); and Smith et al., March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure (6th ed., 2007).

TABLE 1 A² - Moieties HA1

HA2

HA3

HA4

HA5

HA6

HA7

HA8

HA9

HA10

HA11

HA12

HA13

HA14

HA15

HA16

HA17

HA18

HA19

HA20

HA21

HA22

HA23

HA24

TABLE 2 -X²-X¹-A¹ Moieties HB1 —C(═O)—(CH₂)₁₄—C(═O)—OH HB2—C(═O)—(CH₂)₁₆—C(═O)—OH HB3 —C(═O)—(CH₂)₁₈—C(═O)—OH HB4—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB5 —C(═O)—(CH₂)₁₄—C(═O)—O—CH₃ HB6—C(═O)—(CH₂)₁₆—C(═O)—O—CH₃ HB7 —C(═O)—(CH₂)₁₈—C(═O)—O—CH₃ HB8—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—O—CH₃ HB9 —C(═O)—(CH₂)₁₄—CH₃ HB10—C(═O)—(CH₂)₁₆—CH₃ HB11 —C(═O)—(CH₂)₁₈—CH₃ HB12—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—CH₃ HB13—C(═O)—(CH₂)₂—C(═O)—O—(CH₂)₁₅—C(═O)—OH HB14—C(═O)—(CH₂)₂—C(═O)—O—(CH₂)₁₇—C(═O)—OH HB15—C(═O)—(CH₂)₂—C(═O)—O—(CH₂)₁₉—C(═O)—OH HB16—C(═O)—(CH₂)₂—C(═O)—O—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB17—C(═O)—CH₂—NH—C(═O)—(CH₂)₁₄—C(═O)—OH HB18—C(═O)—CH₂—NH—C(═O)—(CH₂)₁₆—C(═O)—OH HB19—C(═O)—CH₂—NH—C(═O)—(CH₂)₁₈—C(═O)—OH HB20—C(═O)—CH₂—NH—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB21—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₄—C(═O)—OH HB22—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₆—C(═O)—OH HB23—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₈—C(═O)—OH HB24—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OHHB25 —C(═O)—O—(CH₂)₁₅—C(═O)—OH HB26 —C(═O)—O—(CH₂)₁₇—C(═O)—OH HB27—C(═O)—O—(CH₂)₁₉—C(═O)—OH HB28 —C(═O)—O—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OHHB29 —C(═O)—NH—(CH₂)₁₅—C(═O)—OH HB30 —C(═O)—NH—(CH₂)₁₇—C(═O)—OH HB31—C(═O)—NH—(CH₂)₁₉—C(═O)—OH HB32 —C(═O)—NH—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OHHB33 —O—(CH₂)₁₅—C(═O)—OH HB34 —O—(CH₂)₁₇—C(═O)—OH HB35—O—(CH₂)₁₉—C(═O)—OH HB36 —O—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB37—NH—(CH₂)₂—O—C(═O)—(CH₂)₁₄—C(═O)—OH HB38—NH—(CH₂)₂—O—C(═O)—(CH₂)₁₆—C(═O)—OH HB39—NH—(CH₂)₂—O—C(═O)—(CH₂)₁₈—C(═O)—OH HB40—NH—(CH₂)₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB41—O—(CH₂)₂—O—C(═O)—(CH₂)₁₄—C(═O)—OH HB42—O—(CH₂)₂—O—C(═O)—(CH₂)₁₆—C(═O)—OH HB43—O—(CH₂)₂—O—C(═O)—(CH₂)₁₈—C(═O)—OH HB44—O—(CH₂)₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB45—NH—CH₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₄—C(═O)—OH HB46—NH—CH₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₆—C(═O)—OH HB47—NH—CH₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₈—C(═O)—OH HB48—NH—CH₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB49—NH—(CH₂)₂—O—C(═O)—(CH₂)₁₄—C(═O)—O—CH₃ HB50—NH—(CH₂)₂—O—C(═O)—(CH₂)₁₆—C(═O)—O—CH₃ HB51—NH—(CH₂)₂—O—C(═O)—(CH₂)₁₈—C(═O)—O—CH₃ HB52—NH—(CH₂)₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—O—CH₃ HB53—CH₂—O—C(═O)—(CH₂)₁₄—C(═O)—OH HB54 —CH₂—O—C(═O)—(CH₂)₁₆—C(═O)—OH HB55—CH₂—O—C(═O)—(CH₂)₁₈—C(═O)—OH HB56—CH₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB57—CH₂—O—C(═O)—(CH₂)₁₄—C(═O)—O—CH₃ HB58 —CH₂—O—C(═O)—(CH₂)₁₆—C(═O)—O—CH₃HB59 —CH₂—O—C(═O)—(CH₂)₁₈—C(═O)—O—CH₃ HB60—CH₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—O—CH₃ HB61—CH₂—O—C(═O)—(CH₂)₁₄—CH₃ HB62 —CH₂—O—C(═O)—(CH₂)₁₆—CH₃ HB63—CH₂—O—C(═O)—(CH₂)₁₈—CH₃ HB64 —CH₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—CH₃ HB65—CH₂—O—C(═O)—CH₂—NH—C(═O)—(CH₂)₁₄—C(═O)—OH HB66—CH₂—O—C(═O)—CH₂—NH—C(═O)—(CH₂)₁₆—C(═O)—OH HB67—CH₂—O—C(═O)—CH₂—NH—C(═O)—(CH₂)₁₈—C(═O)—OH HB68—CH₂—O—C(═O)—CH₂—NH—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB69—CH₂—O—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₄—C(═O)—OH HB70—CH₂—O—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₆—C(═O)—OH HB71—CH₂—O—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₁₈—C(═O)—OH HB72—CH₂—O—C(═O)—(CH₂)₂—C(═O)—O—[(CH₂)₂—O—]₆C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OHHB73 —CH₂—O—C(═O)—O—(CH₂)₁₅—C(═O)—OH HB74—CH₂—O—C(═O)—O—(CH₂)₁₇—C(═O)—OH HB75 —CH₂—O—C(═O)—O—(CH₂)₁₉—C(═O)—OHHB76 —CH₂—O—C(═O)—O—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB77—CH₂—O—C(═O)—NH—(CH₂)₁₅—C(═O)—OH HB78 —CH₂—O—C(═O)—NH—(CH₂)₁₇—C(═O)—OHHB79 —CH₂—O—C(═O)—NH—(CH₂)₁₉—C(═O)—OH HB80—CH₂—O—C(═O)—NH—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB81 ═N—O—(CH₂)₁₅—C(═O)—OHHB82 ═N—O—(CH₂)₁₇—C(═O)—OH HB83 ═N—O—(CH₂)₁₉—C(═O)—OH HB84═N—O—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB85 ═N—NH—(CH₂)₁₅—C(═O)—OH HB86═N—NH—(CH₂)₁₇—C(═O)—OH HB87 ═N—NH—(CH₂)₁₉—C(═O)—OH HB88═N—NH—(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB89═N—O—[(CH₂)₂—O—]₆(CH₂)₁₅—C(═O)—OH HB90 ═N—O—[(CH₂)₂—O—]₆(CH₂)₁₇—C(═O)—OHHB91 ═N—O—[(CH₂)₂—O—]₆(CH₂)₁₉—C(═O)—OH HB92═N—O—[(CH₂)₂—O—]₆(CH₂)₈—CH═CH—(CH₂)₇—C(═O)—OH HB93—C(═O)—CH₂—O—C(═O)—(CH₂)₁₄—C(═O)—OH HB94—C(═O)—CH₂—O—C(═O)—(CH₂)₁₆—C(═O)—OH HB95—C(═O)—CH₂—O—C(═O)—(CH₂)₁₈—C(═O)—OH HB96—C(═O)—CH₂—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB97—C(═O)—CH(CH₃)—O—C(═O)—(CH₂)₁₄—C(═O)—OH HB98—C(═O)—CH(CH₃)—O—C(═O)—(CH₂)₁₆—C(═O)—OH HB99—C(═O)—CH(CH₃)—O—C(═O)—(CH₂)₁₈—C(═O)—OH HB100—C(═O)—CH(CH₃)—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH HB101—C(═O)—(CH₂)₅—O—C(═O)—(CH₂)₁₄—C(═O)—OH HB102—C(═O)—(CH₂)₅—O—C(═O)—(CH₂)₁₆—C(═O)—OH HB103—C(═O)—(CH₂)₅—O—C(═O)—(CH₂)₁₈—C(═O)—OH HB104—C(═O)—(CH₂)₅—O—C(═O)—(CH₂)₇—CH═CH—(CH₂)₇—C(═O)—OH

TABLE 3 Compound No. A²- Moiety -X²-X¹-A¹ Moiety  1-44 HA1 HB1, HB2,HB3, HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15,HB16, HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27,HB28, HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99,HB100, HB101, HB102, HB103, HB104, respectively 45-88 HA2 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively  89-132 HA9 HB1, HB2, HB3, HB4,HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16, HB17,HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28, HB29,HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 133-176 HA12 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 177-220 HA14 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 221-264 HA15 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 265-308 HA16 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 309-352 HA19 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 353-396 HA20 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 397-440 HA21 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 441-484 HA22 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 485-528 HA23 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 529-572 HA24 HB1, HB2, HB3,HB4, HB5, HB6, HB7, HB8, HB9, HB10, HB11, HB12, HB13, HB14, HB15, HB16,HB17, HB18, HB19, HB20, HB21, HB22, HB23, HB24, HB25, HB26, HB27, HB28,HB29, HB30, HB31, HB32, HB93, HB94, HB95, HB96, HB97, HB98, HB99, HB100,HB101, HB102, HB103, HB104, respectively 573-592 HA5 HB33, HB34, HB35,HB36, HB37, HB38, HB39, HB40, HB41, HB42, HB43, HB44, HB45, HB46, HB47,HB48, HB49, HB50, HB51, HB52, respectively 593-612 HA6 HB33, HB34, HB35,HB36, HB37, HB38, HB39, HB40, HB41, HB42, HB43, HB44, HB45, HB46, HB47,HB48, HB49, HB50, HB51, HB52, respectively 613-632 HA7 HB33, HB34, HB35,HB36, HB37, HB38, HB39, HB40, HB41, HB42, HB43, HB44, HB45, HB46, HB47,HB48, HB49, HB50, HB51, HB52, respectively 633-652 HA8 HB33, HB34, HB35,HB36, HB37, HB38, HB39, HB40, HB41, HB42, HB43, HB44, HB45, HB46, HB47,HB48, HB49, HB50, HB51, HB52, respectively 653-672 HA11 HB33, HB34,HB35, HB36, HB37, HB38, HB39, HB40, HB41, HB42, HB43, HB44, HB45, HB46,HB47, HB48, HB49, HB50, HB51, HB52, respectively 673-692 HA17 HB33,HB34, HB35, HB36, HB37, HB38, HB39, HB40, HB41, HB42, HB43, HB44, HB45,HB46, HB47, HB48, HB49, HB50, HB51, HB52, respectively 693-720 HA3 HB53,HB54, HB55, HB56, HB57, HB58, HB59, HB60, HB61, HB62, HB63, HB64, HB65,HB66, HB67, HB68, HB69, HB70, HB71, HB72, HB73, HB74, HB75, HB76, HB77,HB78, HB79, HB80, respectively 721-748 HA4 HB53, HB54, HB55, HB56, HB57,HB58, HB59, HB60, HB61, HB62, HB63, HB64, HB65, HB66, HB67, HB68, HB69,HB70, HB71, HB72, HB73, HB74, HB75, HB76, HB77, HB78, HB79, HB80,respectively 749-760 HA10 HB81, HB82, HB83, HB84, HB85, HB86, HB87,HB88, HB89, HB90, HB91, HB92, respectively 761-772 HA13 HB81, HB82,HB83, HB84, HB85, HB86, HB87, HB88, HB89, HB90, HB91, HB92, respectively773-784 HA18 HB81, HB82, HB83, HB84, HB85, HB86, HB87, HB88, HB89, HB90,HB91, HB92, respectively

Pharmaceutical Compositions

In certain aspects, the compounds of any of the preceding embodimentsmay be formulated into pharmaceutical compositions in any suitablemanner. In general, as compounds for the treatment of cancer, suchpharmaceutical formulations are aqueous formulations suitable forparenteral administration, such as intravenous or intra-arterialadministration.

In at least one aspect, the disclosure provides pharmaceuticalcompositions that include one or more compounds of formula (I)(according to any of the foregoing embodiments) and a protein. In someembodiments, the protein is an albumin or an albumin mimetic. In somesuch embodiments, the protein is human serum albumin (HSA) or a mimeticthereof, i.e., a protein whose sequence is at least 50% equivalent tothat of HSA, or at least 60% equivalent to that of HSA, or at least 70%equivalent to that of HSA, or at least 80% equivalent to that of HSA, orat least 90% equivalent to that of HSA, or at least 95% equivalent tothat of HSA, at least 97% equivalent to that of HSA, at least 99%equivalent to that of HSA. In some embodiments, the protein is humanserum albumin.

In certain embodiments of any of the foregoing embodiments, thepharmaceutical composition also includes a carrier, such as a liquidcarrier. In some embodiments, the carrier includes water. For example,in some such embodiments, water makes up at least 50% by volume, or atleast 60% by volume, or at least 70% by volume, or at least 80% byvolume, or at least 90% by volume, based on the total volume of liquidmaterials in the pharmaceutical composition. The carrier can alsoinclude other liquid ingredients, such as liquid ingredients commonlyincluded in aqueous pharmaceutical formulations for parenteraladministration.

In certain embodiments having an aqueous carrier, the compounds offormula (I) bind non-covalently to the protein in the pharmaceuticalformulation. In some embodiments, the compound of formula (I) and theprotein (e.g., human serum albumin) are non-covalently associated witheach other with a binding constant (K_(b)) of at least 10² M⁻¹, or atleast 10³ M⁻¹, or at least 10⁴ M⁻¹, or at least 10⁵ M⁻¹ at 25° C. in theaqueous composition.

In some embodiments having an aqueous carrier, the compound of formula(I) and the protein are solvated by the carrier. In some suchembodiments, at least 90% by weight, or at least 95% by weight, or atleast 97% by weight, or at least 98% by weight, or at least 99% byweight of the compounds of formula (I) in the composition are boundnon-covalently to the protein with a binding constant (K_(b)) of atleast 10² M⁻¹, or at least 10³ M⁻¹, or at least 10⁴ M⁻¹, or at least 10⁵M⁻¹ at 25° C. in the aqueous composition. In some further suchembodiments, the composition is substantially free of agglomerates ornanoparticles. For example, in some embodiments of any of theaforementioned embodiments, no more than 5% by weight, or no more than4% by weight, or no more than 3% by weight, or no more than 2% byweight, or no more than 1% by weight of the protein-compound (i.e.,non-covalently bound conjugates between the protein and one or morecompounds of formula (I)) in the aqueous composition have a radiusgreater than 7 nm, or a radius greater than 5 nm, or a radius greaterthan 4 nm, as measured by dynamic light scattering.

The compound of formula (I) can have any suitable molar ratio to theprotein in the formulation. For example, in some embodiments of any ofthe foregoing embodiments, the molar ratio of the compound of formula(I) to the protein ranges from 1:10 to 20:1, or from 1:5 to 15:1, orfrom 1:2 to 10:1. In some embodiments of any of the foregoingembodiments, the molar ratio of the compound of formula (I) to theprotein is about 1:1, or is about 2:1, or is about 3:1, or is about 4:1,or is about 5:1, or is about 6:1, or is about 7:1, wherein the term“about,” in this instance means±0.5:1, such that “about 5:1” refers to arange from 4.5:1 to 5.5:1.

In at least one aspect, the disclosure provides pharmaceuticalcompositions that include: a compound, which comprises a cytotoxic drugmoiety and a protein binding moiety; a protein, wherein the protein isan albumin or an albumin mimetic; and a carrier, which comprises water.

In some embodiments, the protein is human serum albumin (HSA) or amimetic thereof, i.e., a protein whose sequence is at least 50%equivalent to that of HSA, or at least 60% equivalent to that of HSA, orat least 70% equivalent to that of HSA, or at least 80% equivalent tothat of HSA, or at least 90% equivalent to that of HSA, or at least 95%equivalent to that of HSA, at least 97% equivalent to that of HSA, atleast 99% equivalent to that of HSA. In some embodiments, the protein ishuman serum albumin.

As noted above, in some embodiments, the carrier includes water. Forexample, in some such embodiments, water makes up at least 50% byvolume, or at least 60% by volume, or at least 70% by volume, or atleast 80% by volume, or at least 90% by volume, based on the totalvolume of liquid materials in the pharmaceutical composition. Thecarrier can also include other liquid ingredients, such as liquidingredients commonly included in aqueous pharmaceutical formulations forparenteral administration.

In certain embodiments, the compounds bind non-covalently to the proteinin the pharmaceutical formulation. In some embodiments, the compound andthe protein (e.g., human serum albumin) are non-covalently associatedwith each other with a binding constant (K_(b)) of at least 10²M⁻¹, orat least 10³M⁻¹, or at least 10⁴M⁻¹, or at least 10⁵M⁻¹ at 25° C. in theaqueous composition.

In some embodiments having an aqueous carrier, the compound and theprotein are solvated by the carrier. In some such embodiments, at least90% by weight, or at least 95% by weight, or at least 97% by weight, orat least 98% by weight, or at least 99% by weight of the compounds offormula (I) in the composition are bound non-covalently to the proteinwith a binding constant (K_(b)) of at least 10² M⁻¹, or at least 10³M⁻¹, or at least 10⁴ M⁻¹, or at least 10⁵ M⁻¹ at 25° C. in the aqueouscomposition. In some further such embodiments, the composition issubstantially free of agglomerates or nanoparticles. For example, insome embodiments of any of the aforementioned embodiments, no more than5% by weight, or no more than 4% by weight, or no more than 3% byweight, or no more than 2% by weight, or no more than 1% by weight ofthe protein-compound (i.e., non-covalently bound conjugates between theprotein and one or more compounds of formula (I)) in the aqueouscomposition have a radius greater than 7 nm, or a radius greater than 5nm, or a radius greater than 4 nm, as measured by dynamic lightscattering.

The compound of formula (I) can have any suitable molar ratio to theprotein in the formulation. For example, in some embodiments of any ofthe foregoing embodiments, the molar ratio of the compound of formula(I) to the protein ranges from 1:10 to 20:1, or from 1:5 to 15:1, orfrom 1:2 to 10:1. In some embodiments of any of the foregoingembodiments, the molar ratio of the compound of formula (I) to theprotein is about 1:1, or is about 2:1, or is about 3:1, or is about 4:1,or is about 5:1, or is about 6:1, or is about 7:1, wherein the term“about,” in this instance means±0.5:1, such that “about 5:1” refers to arange from 4.5:1 to 5.5:1.

The pharmaceutical compositions of any of the foregoing aspects andembodiments can also include certain additional ingredients, such asthose commonly employed in pharmaceutical compositions for parenteraladministration.

Methods and Uses

The compounds or compositions of any of the foregoing embodiments areuseful in the treatment of cancer and related disorders. Therefore,these compounds and compositions can be used for administration to asubject who has or has had a cancerous tumor.

Thus, in certain aspects, the disclosure provides methods of treatingcancer, including administering to a subject a compound or compositionof any of the foregoing aspects and embodiments. In some embodiments,the subject is a human. In some embodiments, the subject is a subject inneed of such treatment, e.g., a human in need of such treatment.

In some aspects, the disclosure provides methods of inducing apoptosisin a cancer cell, including contacting the cancer cell with a compoundor composition of any of the foregoing aspects and embodiments.

In some aspects, the disclosure provides methods of inhibitingproliferation of a cancerous tumor, including contacting the canceroustumor with a compound or composition of any of the foregoing aspects andembodiments.

In some aspects, the disclosure provides uses of a compound orcomposition of any of the foregoing aspects and embodiments as amedicament.

In some aspects, the disclosure provides uses of a compound orcomposition of any of the foregoing aspects and embodiments for treatingcancer.

In some aspects, the disclosure provides uses of a compound of any ofthe foregoing aspects and embodiments in the manufacture of amedicament.

In some aspects, the disclosure provides uses of a compound of any ofthe foregoing aspects and embodiments in the manufacture of a medicamentfor treating cancer.

EXAMPLES

The following examples show certain illustrative embodiments of thecompounds, compositions, and methods disclosed herein. These examplesare not to be taken as limiting in any way. Nor should the examples betaken as expressing any preferred embodiments, or as indicating anydirection for further research.

The examples may use abbreviations for certain common chemicals. Thefollowing abbreviations refer to the compounds indicated.

-   -   DMF=Dimethylformamide    -   DCM=Dichloromethane    -   NMR=Nuclear magnetic resonance    -   HPLC=High-performance liquid chromatography    -   RP-HLPC=Reverse-phase high-performance liquid chromatography    -   LRMS=Liquid chromatography/low-resolution mass spectrometry    -   HRMS=Liquid chromatography/high-resolution mass spectrometry    -   Tips=Triisopropylsilyl    -   DMAP=4-(Dimethylamino)pyridine    -   EDC=1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide    -   THF=Tetrahydrofuran    -   Dipea=N,N-diisopropylethylamine    -   HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium        3-oxide hexafluorophosphate    -   DCC=N,N′-dicyclohexylcarbodiimide    -   HSA=Human serum albumin

Example 1—Mono-Tips Protected C18 Diacid

To a solution of the diacid (10 g) in dry DMF (150 mL) at 60° C. wasadded triisopropylsilyl chloride (6.68 mL). To the stirred solution wasadded dropwise freshly distilled triethylamine (4.35 mL). The reactionwas stirred overnight under nitrogen atmosphere then the solution wascooled to room temperature, filtered and concentrated to dryness.Dichloromethane (150 mL) was added to the solid and the round bottomsonicated. The solid was filtered off and the solvent was removed undervacuum. The residue was purified by column chromatography (2.5% THF inDCM) to give a clear oil. 6.1 g, 41% yield. LRMS—471.32 [M+H]⁺, HRMSTheoretical=471.3864, Observed=471.3861. ¹H NMR (CDCl₃): δ(ppm)1.06-1.09 (m, 21H), 1.2-1.4 (m, 24H), 1.55-1.70 (m, 4H), 2.30-2.40 (m,4H).

Example 2—Paclitaxel-C18 Diacid Conjugate (PTX-FA18)

To a stirring solution of paclitaxel (500 mg) in dry DCM (50 mL) at 0°C. was added DMAP (231 mg). After 5 minutes, EDC (137 mg) was added.After an additional 5 minutes, was added Mono-Tips Protected C18 Diacid(791 mg) and the resulting solution allowed to stir, and warm to roomtemperature overnight. The crude reaction mixture was then transferredto a separatory funnel and washed with water (3×50 mL), saturated NaCl(3×50 mL) and 0.1M HCl (3×50 mL). The organic phase was dried overMgSO₄, and rotovapped to afford a yellowish-white solid. To remove theTIPS protecting group, the solid was taken up in THF and Bu₄NF (1.2 g,0.0046 mol) was added and stirred. After 18 h, excess AMBERLITE ionexchange resin and excess CaCO₃ were added to the reaction mixture.After 1 h, the solution was filtered and the filtrate concentrated to afree-flowing oil. The oil was dissolved in 50 mL DCM, and upon additionof water a white precipitate crashed out. The reaction mixture wasfiltered and transferred to a separatory funnel, where it was extractedwith water (3×50 mL). The organic phase was dried over MgSO₄, filtered,and concentrated to afford a clear/white, glassy solid. 521 mg, 81%yield. MS Theoretical=1147.59 [M−H]⁻, Observed=1148.38. ¹H NMR (CDCl₃):δ(ppm) 0.08-1.10 (m, 21H), 1.2-1.4 (m, 24H), 1.55-1.64 (m, 4H), 1.65-1.7(m, 2H), 1.8-1.95 (m, 4H), 1.95-1.05 (m, 2H), 2.1-2.15 (m, 4H),2.25-2.40 (m, 6H), 2.4-2.5 (m, 2H), 2.5-2.55 (m, 1H), 3.7 (s, 1H),3.75-3.95 (m, 3H), 4.1 (m, 1H), 4.2 (m, 1H), 4.3 (m, 1H), 4.4 (m, 1H),4.7 (m, 1H), 4.95 (m, 1H), 5.5 (m, 1H), 5.7 (m, 1H), 5.8 (m, 1H),6.2-6.3 (m, 2H), 6.8 (m, 1H), 6.97 (m, 1H), 7.05 (m, 1H), 7.3-7.45 (m,5H), 7.45-7.55 (m, 4H), 7.6 (m, 1H), 7.75 (m, 2H), 8.1-8.2 (m, 2H).

Example 3—Paclitaxel-C16 Diacid Conjugate (PTX-FA16)

Hexadecanedioic acid (250 mg), paclitaxel (372 mg), EDC (251 mg) andDMAP (160 mg) were dissolved in DMF (5 mL) and stirred under a nitrogenatmosphere overnight. The reaction mixture was concentrated to dryness,the residue dissolved in chloroform and washed with water (3×20 mL). Theorganic layer was dried Na₂SO₄, filtered and concentrated to dryness.The solid was purified by preparative HPLC to give the conjugate as awhite solid. ¹HNMR, CDCl₃, 8.14 (2H, d), 7.74 (2H, d), 7.62 (1H, t),(7.55-7.30, 9H, m), 6.94 (1H, m), 6.31 (1H, s), 6.27 (1H, t), 5.95 (1H,m), 5.69 (1H, d), 5.51 (1H, m), 4.99 (1H, m) 4.45 (1H, m), 4.32 (1H, m),4.21 (1H, m), 3.82 (1H, m), 2.60-1.00 (51H, m). LRMS, ESI, 1121.03[M−H]⁻.

Example 4—Paclitaxel-C20 Diacid Conjugate (PTX-FA20)

Eicosanedioic acid (160 mg), paclitaxel (200 mg), EDC (135 mg) and DMAP(86 mg) were dissolved in DMF (2 mL), stirred under a nitrogenatmosphere overnight, then concentrated to dryness. The solid waspurified by preparative HPLC (65%-95% ACN) to give the conjugate as awhite solid. ¹HNMR, CDCl₃, 8.14 (2H, d), 7.74 (2H, d), 7.62 (1H, t),(7.56-7.30, 9H, m), 6.95 (1H, m), 6.31 (1H, s), 6.27 (1H, t), 5.96 (1H,m), 5.70 (1H, d), 5.51 (1H, m), 4.99 (1H, m) 4.45 (1H, m), 4.33 (1H, m),4.22 (1H, m), 3.82 (1H, m), 2.60-1.00 (59H, m). LRMS, ESI, 1290.67[M−H+TFA]⁻.

Example 5—Paclitaxel-uC18 Diacid Conjugate (PTX-FAu18)

Octadec-9-enedioic acid (37 mg; 85% trans/15% cis), paclitaxel (200 mg),EDC (45 mg) and DMAP (29 mg) were dissolved in DMF (2 mL), stirred undera nitrogen atmosphere overnight, then concentrated to dryness. The solidwas purified by preparative HPLC (65%-95% ACN) to give the conjugate asa white solid. ¹HNMR, CDCl₃, 8.14 (2H, d), 7.74 (2H, d), 7.62 (1H, t),(7.55-7.30, 9H, m), 7.03 (1H, m), 6.30 (1H, s), 6.27 (1H, t), 5.97 (1H,m), 5.69 (1H, d), 5.52 (1H, m), 5.37 (2H, m), 4.99 (1H, m) 4.45 (1H, m),4.33 (1H, m), 4.21 (1H, m), 3.82 (1H, m), 2.60-1.00 (51H, m). LRMS, ESI,1146.72 [M−H]⁻.

Example 6—Methotrexate-C18 Diacid Conjugate (MTX-FA18)

To a solution of methotrexate (66 mg) in DMF (1 mL) was added Dipea (50μL) followed by HATU (55 mg), after stirring for 3 minutes18-(2-aminoethoxy)-18-oxooctadecanoic acid (57 mg) was added and thereaction stirred overnight under a nitrogen atmosphere. The solution wasconcentrated to dryness and the resulting solid purified by preparativeHPLC (40%-50% ACN) to give the conjugate as a yellow solid. ¹HNMR,DMF-d7, 9.33 (1H, bs), 9.17 (1H, bs), 8.82 (1H, s), 8.13 (1H, m), 7.88(2H, m), 6.91 (2H, m), 4.95 (2H, s), 4.10 (1H, m), 3.33 (3H, s),2.45-2.0 (12H, m), 1.55 (4H, m), 1.27, (24H, m). LRMS, ESI, 792.57[M−H]⁻.

Example 7—Carbonate-Modified Intermediate

To a solution of methyl 18-hydroxyoctadecanoate (320 mg) and pyridine(222 μL) in CHCl₃/DMF (8:2) was slowly added the p-nitrophenylchloroformate (246 mmol). The reaction mixture was stirred under anitrogen atmosphere overnight then transferred to a separatory funneland washed with water (×3). The organic layer was dried with Na₂SO₄,filtered and concentrated to dryness. The solid was purified by columnchromatography (100% CHCl₃) to give a white solid. ¹HNMR, CDCl₃, 8.29(2H, d), 7.39 (2H, d), 4.30 (2H, t), 3.67 (3H, s), 2.31 (2H, t), 1.76(2H, m), 1.70-1.58 (2H, m), 1.42 (3H, t), 1.26 (27H, m). LRMS, ESI,480.47 [M+H]⁺.

Example 8—Doxorubicin-C18 Acid-Ester Carbamate Conjugate (DOX-FAE18-Cm)

To a solution of doxorubicin hydrochloride (50 mg) in dry DMF (1 mL) wasadded triethylamine (30 mL) followed by methyl18-(((4-nitrophenoxy)carbonyl)oxy)octadecanoate (50 mg). The reactionwas stirred under a nitrogen atmosphere overnight then concentrated todryness. The residue was dissolved in chloroform, washed with water (×3)and the organic layer dried with Na₂SO₄, filtered and concentrated todryness. The red solid was purified by column chromatography (10%toluene in DCM) to give a red solid. ¹HNMR, CDCl₃, 8.02 (1H, m), 7.78(1H, m), 7.39 (1H, m), 6.80 (1H, m), 5.50 (1H, m), 5.28 (1H, m), 5.06(1H, m), 4.76 (2H, s), 4.56 (1H, m), 4.20-4.05 (4H, m), 3.98 (2H, m),3.85 (1H, m), 3.67 (3H, s) 3.25 (1H, d), 3.04 (1H, m), 2.98 (1H, d),2.36-1.20 (37H, m). LRMS, ESI, 882.90 [M−H]⁻.

Example 9—Pentafluorophenol Intermediate

To a solution of 18-methoxy-18-oxooctadecanoic acid (250 mg),pentafluorophenol (140 mg), and DMAP (23 mg) in ethyl acetate (10 mL)was added a solution of DCC (157 mg) in ethyl acetate (2 mL). Thereaction was stirred under a nitrogen atmosphere overnight. Theresulting precipitate was removed by filtration and the solvent removedunder reduced pressure to give a white solid. ¹HNMR, CDCl₃, 3.67 (3H,s), 2.31 (2H, t), 1.78 (2H, m), 1.62 (2H, m), 1.42 (2H, m), 1.26 (22H,m). LRMS, ESI, 517.40 [M+Na]⁺.

Example 10—Amino Acid-Modified Intermediate

A solution of 1-methyl 18-(perfluorophenyl) octadecanedioate (250 mg),6-aminohexanoic acid (66 mg) and triethylamine (141 μL) in DMF (5 mL)was heated at 60° C. with stirring under a nitrogen atmosphere for 48hrs. The reaction mixture was cooled and concentrated under reducedpressure. The residue was dissolved in chloroform and washed with 10%HCl (×3), the organic layer was dried with Na₂SO₄, filtered andconcentrated to dryness to give a tan solid. ¹HNMR, CDCl₃, 7.72 (1H, m),3.64 (3H, s), 3.14 (2H, m), 2.40-2.20 (4H, m), 2.14 (2H, t), 1.60-1.40(8H, m), 1.26 (26H, m). LRMS, ESI, 442.56 [M+H]⁺.

Example 11—Paclitaxel-C18 Diacid Conjugate+Amino Acid Spacer(PTX-FAE18-Aa)

To a solution of 6-(18-methoxy-18-oxooctadecanamido)hexanoic acid (103mg), Paclitaxel (299 mg) and DMAP (43 mg) in DMF (2 mL) was added EDC(67 mg). The reaction mixture was stirred under a nitrogen atmosphereovernight then concentrated to dryness. The residue was dissolved inchloroform and washed with 0.1M HCl (3×). The organic layer was driedwith Na₂SO₄, filtered and concentrated to dryness. The solid waspurified by preparative HPLC (65%-95% ACN) to give the conjugate as awhite solid. ¹HNMR, CDCl₃, 8.14 (2H, d), 7.75 (2H, d), 7.62 (1H, m),(7.55-7.30, 10H, m), 7.11 (1H, m), 6.30 (1H, s), 6.23 (1H, m), 5.95 (1H,m), 5.68 (1H, m), 5.50 (1H, m), 4.98 (1H, m) 4.45 (1H, m), 4.32 (1H, m),4.21 (1H, m), 3.82 (1H, m), 3.67 (3H, s), 3.18 (2H, m), 2.60-1.00 (62H,m). LRMS, ESI, 1312.52 [M−H+Cl]⁻.

Example 12—Camptothecin-C18 Diacid Conjugate (CPT-FA18)

Camptothecin was stirred with the octadecanedioic acid in DMF, alongwith EDC and DMAP in excess. After reaction was complete, reactionsolution was concentrated, brought up in DCM, then washed 2×1M HCl and1× H₂O. The organic layer was dried over MgSO₄, filtered andconcentrated. Crude FA-CPT was purified first by flash chromatographyusing a gradient elution (100% DCM→10% methanol in DCM); then purifiedby prep RP-HPLC using a 70-90% acetonitrile in water+0.1% TFA gradient,monitoring at 260 nm. The purified product was lyophilized to remove theHPLC solvents, leaving a yellow-tinted powder. ¹HNMR, CDCl₃, 8.41 (s,1H), 8.23 (d, 1H, J=8.4 Hz), 7.95 (d, 1H, J=8.1 Hz), 7.84 (t, 1H, J=8.5Hz), 7.68 (t, 1H, 7.1 Hz), 7.26 (s, 1H), 5.68 (d, 1H, J=17.2 Hz), 5.42(d, 1H, J=17.2 Hz), 5.31 (s, 2H), 2.49 (m, 2H), 2.35 (t, 2H, J=8.0 Hz),2.29 (m, 1H), 2.17 (m, 1H), 1.70-1.59 (m, 4H), 1.39-1.11 (m, 24H), 0.98(t, 3H, 7.4 HZ). LRMS, ESI, 644.83 [M−H]⁻.

Example 13—Ixabepilone-C18 Diacid Conjugate (IXB-FA18)

To a stirring solution of ixabepilone (500 mg) in dry DCM (50 mL) at 0°C. is added DMAP (231 mg). After 5 minutes, EDC (137 mg) is added. Afteran additional 5 minutes, is added Mono-Tips Protected C18 Diacid (791mg) and the resulting solution allowed to stir, and warm to roomtemperature overnight. The crude reaction mixture is then transferred toa separatory funnel and washed with water (3×50 mL), saturated NaCl(3×50 mL) and 0.1M HCl (3×50 mL). The organic phase is dried over MgSO₄,and rotovapped to afford a yellowish-white solid. To remove the TIPSprotecting group, the solid is taken up in THF and Bu₄NF (1.2 g) isadded and stirred. After 18 h, excess AMBERLITE ion exchange resin andexcess CaCO₃ are added to the reaction mixture. After 1 h, the solutionis filtered and the filtrate is concentrated to a free-flowing oil. Theoil is dissolved in 50 mL DCM, and upon addition of water a whiteprecipitate forms. The reaction mixture is filtered and transferred to aseparatory funnel, where it is extracted with water (3×50 mL). Theorganic phase is dried over MgSO₄, filtered, and is concentrated toafford a solid.

MS—Theoretical=803.16 [M−H]⁻.

Example 14—Complexation of PTX-FA to Human Serum Albumin (PTX-FA-HSA)

Human serum albumin (HSA) was dissolved in distilled water to yield a 4%(w/v) solution (1000 uL, 7.19×10⁻⁴ M). To this was rapidly added a stocksolution of PTX-FA (100 uL, 0.0144 M). The resulting solution wassonicated for 10 seconds, snap frozen, and lyophilized. The lyophilizedpowder was resuspended in 1.0 mL 1×DPBS.

Example 15—Data and Results: In Vivo Efficacy

Equivalent doses (240 mg/kg with respect to paclitaxel content) ofPTX-FA-HSA was given to tumor-burdened (HT-1080) nu/nu mice (n=3), andthe effect of tumor growth was monitored over the course of two weeks,administered intravenously (for PTX-FA-HSA). Saline (n=2) wasadministered as a negative control. Table 4 below shows the averagerelative tumor volume (relative to day 0).

TABLE 4 Day PTX-FA-HSA Post- 240 mg/kg IV Saline Injection (n = 3) (n =2) 0 1.00 1.00 1 1.05 0.73 3 1.15 0.81 5 1.58 2.23 7 3.04 6.17 14 8.6822.21

Further, in vivo analysis of protein-bound PTX-HA in HT-1080fibrosarcoma xenografts was carried out to measure appropriate doses andsurvival. Results are shown in Table 5. Doses all reported with respectto paclitaxel content of treatment. Minimum Effective Dose (MED) isdefined as lowest dose necessary to extend survival beyond mediansurvival of non-treated (saline) animals. Maximum Effective Dose (MTD)is defined as highest dose resulting in less than 10% weight loss.Therapeutic Index (TI) is defined as the ratio of MTD to MED. MedianSurvival is defined as the time for each cohort to drop below 50%survival. N=6 for all groups. Administration was carried out byintravenously.

TABLE 5 HSA-Bound Parameter PTX-FA Saline MED 5 mg/kg — MTD >250 mg/kg —TI >50 — Median Survival >70 days 25 days

Example 16—Particle Size by Dynamic Light Scattering

Multiple formulations of PTX-FA and HSA were prepared according to themethod of Example 14, and varying the molar ratio of PTX-FA to HSA. Foreach formulation, a particle size analysis was carried out using dynamiclight scattering. Table 6 shows the average radius of the particles andthe percentage of particles (by mass percent) in the formulation havingthat respective radius. FIG. 2 shows a micrograph from a cryo-TEM(cryogenic transmission electron microscopy) at liquid nitrogentemperatures of a solution where the molar ratio of PTX-FA to HSA is1:1. The cryo-TEM results show that the solution contains no aggregates.

TABLE 6 Percentage Molar Ratio Particle Radius of Particles (PTX-FA:HSA)(nm) (mass %) 1:1 3.4 99.2 2:1 3.5 96.1 3:1 3.6 95.6 Free HSA 3.5 99.8

Example 17—PTX-FA18 Binding to HSA

Using a dialysis-based protein binding assay according to the methoddescribed in Cohen, Plasma Protein-Binding Methods in Drug Discovery inOPTIMIZATION IN DRUG DISCOVERY: IN VITRO METHODS, Yan & Caldwell, eds.,pp. 111-122 (Humana Press, Totowa, N.J., 2004), which is herebyincorporated by reference as though set forth herein in its entirety.The percentage of PTX-FA18 and the percentage of octadecanedioic acid(C18 Diacid) bound to the protein at different concentrations wasmeasured. The results are shown in Table 7.

TABLE 7 Concentration % bound to protein % bound to protein (μM) PTXFAC18 Diacid 20.0 100.0 97.4 10.0 100.0 100.0 3.0 99.7 99.8 1.5 99.5 99.1

Example 18—In Vitro Cytotoxicity

General Cell Culture Methods:

HeLa cells were obtained from ATCC and incubated at 37° C. at 5% CO₂using DMEM supplemented with 10% FBS, and 1× of sodium pyruvate,non-essential amino acids, L-glutamine and antibiotics.

Cell Viability Experiments:

Cytotoxicity of compounds was evaluated using the CellTiter Blue assay(Promega). HeLa cells were plated in 96-well plates, at a density of3000 cells/well 1 day before treatment. Treatments were prepared as1000× serial stock dilutions in DMSO, then diluted into media for 1×,0.1% DMSO treatment solutions. Plating media was removed, thentreatments were added to the wells. After 3 days, the media was removedand replaced with 100 uL complete DMEM without phenol red. Then 20 uL ofCellTiter Blue reagent was added, and the plates incubated for two hoursat 37° C. Fluorescence was measured at 590 nm with excitation at 560 nm.To determine viability, average background fluorescence was subtractedfrom average fluorescence readings of the experimental wells (threewells per treatment concentration). Viability was calculated as theaverage background-subtracted signal in a well compared to that of anegative control well (treatment with 0.1% DMSO/media).

Determining IC₅₀:

Viabilities were fit in GraphPad Prism using a non-linear,dose-dependent inhibition curve. The IC₅₀ numbers given in the tablereflect the concentration at which the cell death is 50% of the maximumresponse.

Results:

Table 8 shows IC₅₀ (measured as described above) values for variousHSA/Drug-FA conjugates that were synthesized in the above examples.

TABLE 8 Compound IC₅₀ PTX-FA18 64.42 nM PTX-FA16 137.0 nM PTX-FA20 27.8nM PTX-FAu18 40.06 nM PTX-FAE18-Aa 2.329 uM* MTX-FA18 3.009 uM CPT-FA1810.97 uM Reported IC₅₀ values are averages of at least 3 replicates,except * which is one experiment.

The invention claimed is:
 1. A compound of formula (1)A¹-X¹—X²-A²  (I) wherein: A¹ is a carboxylic acid group, a carboxylateanion, or a carboxylate ester of the formula —COOR^(a), wherein R^(a) isan alkyl or an alkoxylate group; A² is a paclitaxel moiety; X¹ is C₁₂₋₂₂straight-chain alkylene or C₁₂₋₂₂ straight-chain alkenylene, wherein A¹is separated from X² by at least 6 carbon atoms; and X² is an organicgroup, wherein the organic group comprises one or more moieties selectedfrom the group consisting of: —C(═O)—, —O—C(═O)—, —NH—C(═O)—, one ormore units formed from alkylene glycols, one or more units formed fromalkanol amines, one or more units formed from amino acids, one or moreunits formed from hydroxyl acids, and combinations thereof, and whereinthe organic group comprises from 1 to 100 carbon atoms.
 2. The compoundof claim 1, wherein A¹ is a carboxylic acid group.
 3. The compound ofclaim 1, wherein the paclitaxel moiety is a moiety of the formula:


4. The compound of claim 1, wherein X¹ is C₁₂₋₂₂ straight-chain alkylenegroup.
 5. The compound of claim 1, wherein X² is —C(═O)—.
 6. Apharmaceutical composition comprising: a compound of claim 1; and aprotein, wherein the protein is human serum albumin or a protein whosesequence is at least 50% equivalent to that of human serum albumin. 7.The pharmaceutical composition of claim 6, wherein the protein is humanserum albumin.
 8. The pharmaceutical composition of claim 6, furthercomprising a carrier.
 9. The pharmaceutical composition of claim 8,wherein the carrier comprises water.
 10. The pharmaceutical compositionof claim 9, wherein the compound and the protein are non-covalentlyassociated with each other with a binding constant (K_(b)) of at least10² M⁻¹.
 11. The pharmaceutical composition of claim 8, wherein thecompound and the protein are solvated by the carrier.
 12. A method oftreating cancer, comprising: administering to a subject a composition ofclaim
 6. 13. The compound of claim 1, wherein X¹ is C₁₄₋₂₂straight-chain alkylene.
 14. The compound of claim 1, wherein X¹ isC₁₄₋₂₂ straight-chain alkenylene.
 15. A pharmaceutical compositioncomprising: a compound of claim 13; and a protein, wherein the proteinis human serum albumin or a protein whose sequence is at least 50%equivalent to that of human serum albumin.
 16. A pharmaceuticalcomposition comprising: a compound of formula (1)A¹-X¹—X²-A²  (I) wherein: A¹ is a carboxylic acid group, a carboxylateanion, or a carboxylate ester; A² is a paclitaxel moiety; X¹ is C₁₂₋₂₂hydrocarbylene, wherein A¹ is separated from X² by at least 6 carbonatoms; and X² is an organic group, wherein the organic group comprisesone or more moieties selected from the group consisting of: —C(═O)—,—O—C(═O)—, —NH—C(═O)—, one or more units formed from alkylene glycols,one or more units formed from alkanol amines, one or more units formedfrom amino acids, one or more units formed from hydroxyl acids, andcombinations thereof, and wherein the organic group comprises from 1 to100 carbon atoms; and a protein, wherein the protein is human serumalbumin or a protein whose sequence is at least 50% equivalent to thatof human serum albumin.